Decarboxylase Test: Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, Limitations, and Keynotes

Principle of Decarboxylase-Dihydrolase Test

Lysine, ornithine, and arginine are the most common amino acids which use to detect an organism’s ability to decarboxylate or hydrolyze an amino acid in a decarboxylase medium, forming an amine that produces an alkaline pH. The basal medium for the decarboxylase test is usually Moller’s formula and contains constituents like meat peptones and beef extract, which supply nitrogenous nutrients to support bacterial growth. Glucose is a fermentable carbohydrate. The two pH indicators are bromocresol purple and cresol red. Pyridoxal is an enzyme cofactor that enhances decarboxylase activity. Arginine, lysine, and ornithine are amino acids that are singly added to the basal medium. It helps to detect the production of enzymes that decarboxylate or hydrolyze these substrates.

When an organism in the medium ferments glucose, the production of acids occurs which lowers the pH, resulting in a color change from purple to yellow. If decarboxylation or hydrolysis of the amino acid occurs in response to the acid pH, alkaline end product (amines) formation happens which results in the medium reverting to its original color (purple). When the organism does not ferment glucose, the medium does not turn yellow. In this scenario, the test can still be performed. But it is important to include a control without amino acids for comparison.

Decarboxylation of lysine yields cadaverine, decarboxylation of ornithine yields putrescine, and decarboxylation of arginine yields agmatine. And it is hydrolyzed by a dihydrolase to form putrescine. In another reaction, arginine dihydrolase converts arginine to citrulline, which is converted to ornithine and then to putrescine. Since decarboxylation is an anaerobic reaction, the contents of each tube must be sealed with oil.

Composition of the Decarboxylase Medium

Moeller Decarboxylase Base:

Peptone: 5.0gm

Beef Extract: 5.0gm

Glucose: 0.5gm

Bromcresol Purple: 0.5gm

Cresol Red: 5.0mg

Pyridoxal: 5.0mg

Distilled water: 1000 ml

Additionally,

Arginine: 10.0gm for Moeller Arginine Decarboxylase medium

Similarly, Lysine:10.0 gram for Moeller Lysine Decarboxylase medium

And Ornithine: 10.0 gram for Moeller Ornithine  Decarboxylase medium

Final pH 6.0 +/- 0.2 at 25ºC.

Requirements for Decarboxylase Test

• Moeller basal Decarboxylase medium
• Moeller Lysine Decarboxylase medium
• Moeller Ornithine Decarboxylase medium
• Moeller Arginine Decarboxylase medium
• Test organism
• Bunsen burner
• Inoculating loop or wire or sterile stick
• Incubator
• Mineral oil or melted petroleum jelly, Vaspar, or paraffin,
• Control strains: Klebsiella pneumoniae ATCC 13883 and Enterobacter cloacae ATCC 23355

Procedure of Decarboxylase Test

1. Inoculate each broth being tested with one or two colonies from an 18- to 24-hour culture.
2. For non-glucose-fermenting, gram-negative rods a. Include the control tube b. Inoculate the tubes heavily i.e. no. 8 McFarland turbidity standard. For Enterobacteriaceae, the control tube is not needed, since all strains ferment glucose.
3. For gram-positive cocci, the control tube is usually not needed.
4. Overlay the inoculated tubes with approximately 1 ml of mineral oil or a 4-mm plug of mineral oil, being careful to cover the broth layer entirely without introducing air.
5. Tighten the caps on the tubes.
6. Incubate aerobically at 35°C for at least 18 hours and up to 7 days, and observe daily for purple color.

Result and Interpretation of Decarboxylase Test

Positive test:  turbid purple to faded-out yellow-purple color (alkaline)

Negative test: bright clear yellow color (acid) or no change (non-fermenting rods)

The control tube remains its original color or turns yellow.

Note: An alkaline or purple color in the control invalidates the test.

Control strains

Positive

• Lysine: Klebsiella pneumoniae
• Ornithine: Enterobacter cloacae
• Arginine: Enterobacter cloacae

Negative

• Lysine: Enterobacter cloacae
• Ornithine: Klebsiella pneumoniae
• Arginine: Klebsiella pneumoniae

Uses of Decarboxylase Test

1. Plesiomonas is positive for lysine, arginine, and ornithine, which separates it from Vibrio and Aeromonas, whose results are variable with each species but are not positive for all three decarboxylases.
2. Staphylococcus lugdunensis is the only Staphylococcus that is pyrrolidinyl-bnaphthylamide (PYR) and ornithine positive.
3. Arginine is useful in the identification of Enterococcus to the species level; Enterococcus avium is arginine negative, but Enterococcus faecalis and Enterococcus faecium are arginine positive.
4. Stenotrophomonas maltophilia and Burkholderia cepacia are among the few non-glucose-fermenting, gram-negative rods that are lysine positive. Among the polymyxin B-resistant non-fermenters, Burkholderia mallei and Burkholderia pseudomallei are arginine positive.
5. Lysine Decarboxylase  Test (LDC): To assist in the identification of Salmonellae (positive) and Shigellae (negative). Bacteria that are Lysine Decarboxylase positive are:

• Escherichia coli
• Klebsiella pneumoniae
• Serratia marcescens
• Vibrio cholerae
• Vibrio Parahemolyticus
• Salmonella Typhi and Most other salmonellae species (except Salmonella Paratyphi A).

Limitations of  Decarboxylase Test

1. Test interpretation should not be made prior to 18 to 24 hours of incubation. Earlier interpretation may lead to erroneous results. Glucose fermentation occurs within the first 10 to 12 hours of incubation. Fermentation produces an acidic environment which results in yellow color development. The production of decarboxylase enzymes will not be induced until the acidic state has been established.
2. If two layers of different colors appear, shake the tube gently before interpreting the reaction.
3. Non-glucose-fermenting microorganisms may display weak decarboxylase activity, thereby resulting in an insufficient production of amines necessary to convert the pH indicator system. Some non-fermenters, however, will produce sufficient amines to result in a deeper purple color than in an uninoculated tube of the basal medium.
4. A gray color may indicate a reduction of the indicator, rather than the production of alkaline end products. To aid in reading the reaction, add additional bromocresol purple.
5. Nonfermenting bacteria that are arginine positive must be lysine and ornithine negative.

Mucoid Lactose Fermenting Colony of Klebsiella Pneumoniae on MacConkey agar

Biochemical Reactions of Klebsiella oxytoca in MIU agar, TSI, and Citrate medium

Note: Klebsiella oxytoca is indole positive while K. pneumoniae is indole negative.

Further Readings

1. Lynae S. Carcia, Second Edition update, Clinical Microbiology Procedures Handbook
2. Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
3. B.D. Skerman, A guide to the identification of the genera of bacteria, The Williams & Wilkins Co., Baltimore, MD, (1967)
4. Cowan and Steel’s, manual for the identification of medical bacteria